Crossing a tissue specific RNAi strain: how to check the genome?

Dear members of WormBase,

Recently I tried to cross a tissue-specific RNAi strain (that possesses rde-1 mutation to make the worm not susceptible to RNAi, but the wild-type
copy of it is present in the form of a construct and it’s expressed under the promoter of a specific tissue) with another mutant. The problem is that
by sequencing I can’t differentiate whether my new mutant has the rde-1 mutation or not, because of the abundance of the wild-type gene. Is there a
way to detect the mutation in the genome? Would be very grateful for any advice!

Thanks you in advance!

How are you designing your sequencing primers? The tissue specific rescuing construct uses the rde-1 cDNA (not genomic) right?

Construct your strain with an rde-1 balancer so you don’t need to check the allele molecularly. unc-42 sma-1 double works fine to balance rde-1, and I have used it for this purpose before.

What Reiner said.

Another balancer option you might consider is nT1 [qIs51]; the dominant gfp marker can be really useful in your strain constructions, and you can be a little sloppier about monitoring generations because there’s no real risk of recombination (not that the risk with unc-42 sma-1 is big). Not useful if you’re building with something on LGIV or LGV, but otherwise it’ll save you time and trouble.

Hillel’s right of course. Mine included let-60(n1046) on IV, so I wanted to avoid nT1g. But nT1g is far easier if you don’t have another factor on IV (rde-1 is on V)

Dear all, thank you very much for the useful replies. :slight_smile: Dear Snug, I actually didn’t pay attention to the regions that I used to design the primers. Hopefully that will sort out my problem. Thanks :wink: